Causal effects of hypertensive disorders of pregnancy on future gynecologic tumors: A two‐sample Mendelian randomization study

Abstract Background Numerous observational studies have investigated the potential link between hypertensive disorders of pregnancy (HDPs) and the subsequent risks of gynecologic tumors, yet the findings have been inconsistent. In this study, we utilized Mendelian randomization (MR) approach to assess the influence of HDPs on the future risks of ovarian, cervical, endometrial, and breast cancer and uterine fibroids, controlling for confounding factors. Methods The genome‐wide association studies (GWAS) summary data relevant to HDPs was obtained from the FinnGen databases (10,736 cases and 136,325 controls). Gynecologic tumor outcomes were extracted from the IEU Open GWAS project and UK Biobank (47,690 cases and 1, 092,073 controls). The inverse variance weighted (IVW) approach was selected as the principal method for MR analysis, supplemented by MR‐Egger, weighted median, weighted model, simple model methods, MR pleiotropy residual sum and outlier (MR‐PRESSO) test, and leave‐one‐out method. Multivariate MR (MVMR) analysis was conducted after adjusting systolic blood pressure (SBP), body mass index (BMI) and type 2 diabetes mellitus (T2DM). Results Our univariate MR analysis (UVMR) results revealed no significant relationship between HDPs and the risks of ovarian cancer (odds ratio [OR] = 0.924, p = 0.360), cervical cancer (OR = 1.230, p = 0.738), endometrial cancer (OR = 1.006, p = 0.949), uterine fibroids (OR = 1.155, p = 0.158), and breast cancer (OR = 0.792, p = 0.241) by IVW test. Similar results were observed in gestational hypertension and preeclampsia/eclampsia. Additionally, our study detected neither heterogeneity nor pleiotropy. MVMR analysis also provided no evidence of a causal association between HDPs and common gynecologic tumors after adjusting SBP, BMI, and T2DM. Conclusion We discovered no causal relationship between HDPs and ovarian, cervical, endometrial, breast cancer, and uterine fibroids in European populations. However, present analysis did not explore the effect of HDPs on the subtypes of gynecologic tumors across varied ethnic populations, which may require additional research.


| INTRODUCTION
Hypertensive disorders in pregnancy (HDPs), characterized by the onset of elevated blood pressure during gestation, affect an estimated 4.1%-19.4% of pregnancies worldwide. 1As the second biggest cause of maternal death, HDPs also pose a substantial risk to newborn health. 2 HDPs can be categorized into three specific diagnoses: preeclampsia or eclampsia, gestational hypertension, and any of these conditions occurring concurrently with chronic hypertension. 3In addition to the direct impact on maternal and fetal health, HDPs are linked to considerable alterations in future disease risk for the mother.Numerous systematic reviews and meta-analyses have conclusively demonstrated an elevated future cardiovascular disease risk among women who have experienced HDPs. 4,5nsufficient knowledge exists about the correlation between HDPs and the risk of cancer.Most prior research has largely focused on exploring the association between preeclampsia and breast cancer, often yielding inconsistent results. 6,7A recent meta-analysis failed to establish a significant link between maternal breast cancer susceptibility and either preeclampsia or gestational hypertension. 8owever, findings from later studies have been inconsistent.A comprehensive cohort investigation reported a diminished risk of breast cancer in women with a history of HDPs. 9 Several other researches have echoed this finding, suggesting a risk reduction in the range of 10%-20%. 6,7,10evertheless, the potential relationship between HDPs and other gynecologic tumors, such as ovarian, cervical, and endometrial cancer, as well as uterine fibroids, is poorly understood.Current research on these gynecologic tumors has yielded inconsistent outcomes. 11,12Evidence from the European Prospective Investigation into Cancer (EPIC)-Norfolk cohort suggested no association between HDPs and future risk of ovarian, endometrial, and breast cancers. 11Another nationwide cohort study revealed that a history of HDPs was significantly related to decreased incidence of breast cancer and with increased incidence of endometrial cancer. 12Furthermore, the criteria for identifying HDPs varied across research.Powell et al. considered individuals with reports of gestational hypertension and/or preeclampsia as having HDPs. 9By contrast, Goldberg et al. defined women with any self-reported preeclampsia, eclampsia, or high blood pressure during pregnancy as those with a history of HDPs. 13Unquestionably, determining the causal association between HDPs and future gynecologic tumors may have far-reaching influence on cancer prevention strategies.Given the potential for residual confounders, the infeasibility of randomized controlled trials (RCTs), and the scarcity of large cohort studies, innovative methodologies are urgently required to determine causality.
Mendelian randomization (MR) serves as a powerful method for establishing causal inference by utilizing single nucleotide polymorphisms (SNPs) as instrumental variables (IVs). 14Analogous to treatment randomization in clinical trials, genetic variants are assigned randomly during gamete formation and conception, uninfluenced by external factors. 15This results in efficacious randomization of high or low genetic risks for diseases, thus minimizing the possibility of confusion and reverse causality, akin to RCTs.Summary data from genome-wide association studies (GWAS) are more readily available and typically extensive for two-sample MR analysis, which serves to strengthen genetic interpretation of IVs on exposure and enhances the precision and dependability of analysis outcomes. 16Consequently, this study aims to perform a two-sample MR analysis utilizing pooled data from GWAS to explore the relationship between HDPs and the risks of ovarian, cervical, endometrial, and breast cancer, as well as uterine fibroids.

| Study design and data sources
The present study used a two-sample MR analysis to ascertain the causal relationship between exposures and outcomes.For the causal predictions produced from the two-sample MR analysis to hold validity, the SNPs utilized in MR must meet three essential prerequisites: (1) the SNPs were associated with HDPs, gestational hypertension, or preeclampsia/eclampsia; (2) the SNPs were not linked to confounders; (3) the SNPs impacted the outcomes solely via the exposure factors.The MR study design was shown subtypes of gynecologic tumors across varied ethnic populations, which may require additional research.

K E Y W O R D S
breast cancer, endometrial cancer, hypertensive disorders in pregnancy, Mendelian randomization, preeclampsia in Figure 1.The present research was based on publicly accessible summary data from large GWAS, which were from European descent.The ethical approval of all included GWAS data has been accomplished and approved by the Ethics Committee.As the present study only extracted data from GWAS databases for analysis, an ethical review was not necessitated.
The study encompassed three exposure variables: HDPs in aggregate and its two subcategories, preeclampsia/eclampsia and gestational hypertension.The estimates for genetic connections were derived from the sixth data dissemination of GWAS summary data by the FinnGen consortium, which was made available on January 24,2022.The dataset contained HDPs (10,736 cases and 136,325 controls), gestational hypertension (5240 cases and 136,325 controls), and preeclampsia/eclampsia (4743 cases and 136,325 controls).The average age at the initial event ranged from 29.06 to 30.08 years. 17y consulting the PhenoScanner database (http:// www.pheno scann er.medsc hl.cam.ac.uk/ ), we considered systolic blood pressure (SBP), body mass index (BMI), and type 2 diabetes mellitus (T2DM) as potential confounders based on their known or plausible effects on HDPs. 17,18The summary statistics for SBP (422,713 European ancestry individuals), 19 BMI (407, 609 British ancestry individuals), 20 and T2DM (468,298 European ancestry individuals) 21 were obtained from the publications based on the UK Biobank.
The GWAS summary data relevant to ovarian cancer, which is part of the Ovarian Cancer Association Consortium (OCAC), was procured from the IEU Open GWAS project (https:// gwas.mrcieu.ac.uk/ ), encompassing 25,509 cases and 40,941 controls.The summary data on cervical cancer (258 cases and 247,282 controls), uterine fibroids (7187 cases and 240,353 controls), and breast cancer (1830 cases and 454,518 controls) were obtained from results of the GWAS in the UK Biobank. 22SNPs associated with endometrial cancer were sourced from the endometrial GWAS data, which collectively included 12,906 cases and 108,979 controls (Table 1). 23

| Instrumental variable selection
IVs were selected on the basis of their correlation with each exposure at a threshold of p < 5 × 10 −8 .We implemented linkage disequilibrium (LD) using European ancestry reference data (1000 Genomes Project, r 2 = 0.001, strand alignment = 10,000 kb).For preeclampsia/eclampsia, the absence of SNPs at this threshold led us to escalate the p threshold tenfold until a variant was attainable, leading to a threshold of p < 5 × 10 −6 . 17Pleiotropy identification for each selected SNP was performed using Phenoscanner (http:// www.pheno scann er.medsc hl.cam.ac.uk/ ).We included pleiotropic SNPs, but subsequently excluded them if horizontal pleiotropy was detected in sensitivity analysis.To evaluate the potency of the chosen IVs in explaining exposure traits, we computed F-statistic.IVs with an F-statistic less than 10 were excluded. 24Additionally, we harmonized the exposures and outcomes to exclude palindromic SNPs and confirm the uniformity of the effect alleles.Palindromic SNPs were defined as those possessing ambiguous minor allele frequencies falling between >0.45 and <0.55 and were subsequently removed from our dataset. 25,26Details of the included SNPs were presented in Tables S1-S3.

| MR analyses
Univariate MR analysis (UVMR) utilized genetic variants as IVs to assess the causal impact of HDPs on common gynecologic tumors.We selected the inverse variance weighted (IVW) approach as the principal methodology.IVW was with multiplicative random effects whenever a minimum of four SNPs were present. 27When only two or three SNPs were available, a fixed-effects IVW approach was used.To prevent the impact of undetectable and unknown confounders, supplementary analyses, including MR-Egger, 28 weighted median, 29 weighted mode, 30 and simple mode, 30 were conducted.All analyses underwent adjustment through the Bonferroni method. 31In order to tackle multiple testing concerns, we adopted a conservative Bonferroni-corrected p-value <0.003 (0.05/15) via the IVW method to suggest statistical significance, as we examined the relationship between three HDPs-associated traits and five gynecologic tumors.Results with p-value >0.003 but less than 0.05 were considered as potential causal relationships. 32,33To adjust for potential confounders, we executed multivariable MR (MVMR) analysis by incorporating SBP, BMI, and T2DM.The multivariable IVW, multivariable MR-Egger, and multivariable median methods were used. 34

| Sensitivity analyses
We used the Cochran Q statistic to estimate the heterogeneity in IVW analysis (p < 0.05 was considered significant).
To validate the existence of horizontal pleiotropy and evaluate the potential pleiotropic impacts of genetic instruments on the outcomes, we used MR-Egger regression (p < 0.05 was considered significant). 28MR pleiotropy residual sum and outlier (MR-PRESSO) test provided an additional sensitivity assessment.This method allowed the identification and removal of outliers in the MR summary data related to horizontal pleiotropy, and corrected horizontal pleiotropy.Furthermore, MR-PRESSO test enabled the comparison of MR analysis results pre-and post-correction (p < 0.05 was considered significant). 35For sensitivity analysis, we used the leave-one-out method to determine the impact of single SNPs on the overall findings.All statistical analyses were performed in R software 4.3.1 using the "TwoSampleMR", "MVMR", and "MR-PRESSO" packages.

| HDPs and common gynecologic tumors
In the UVMR analysis, three SNPs were obtained from GWAS summary data of HDPs after excluding palindromic SNPs.2).No significant heterogeneity or horizontal pleiotropy was detected.The scatter plots of were provided in Figure S1.The leave-oneout sensitivity analysis revealed that the overall results were not affected by any individual SNP (Figure S2).MR-PRESSO test was not conducted as it necessitates more than three SNPs for analysis (Table 2).

| Preeclampsia/eclampsia and common gynecologic tumors
In the UVMR analysis, 17, 18, 17, 18, and 18 SNPs were eventually obtained as the IVs for preeclampsia/eclampsia to evaluate the relationships with ovarian cancer, cervical cancer, endometrial cancer, uterine fibroids, and  5).This finding suggested that the IVs affected the uterine fibroids risk exclusively through their effect on pre-eclampsia/eclampsia.The scatter plots were presented in Figure S5.Additionally, leave-one-out analysis also did not identify any abnormal SNPs (Figure S6).MVMR analysis revealed that preeclampsia/eclampsia showed no significant association with ovarian cancer, cervical cancer, endometrial cancer, uterine fibroids, and breast cancer after adjusting for SBP, BMI, and T2DM (all p > 0.05).Similar results were obtained through MVMR MR-Egger method.Nevertheless, the MVMR median method showed a potential association between preeclampsia/eclampsia and ovarian cancer (OR [95% CI] = 0.918 [0.855-0.985];p = 0.018) after adjusting for SBP (Table S5).

| DISCUSSION
By using genetic data, we explored the relationship of HDPs on risk of five common gynecologic tumors.The results revealed that there is no strong evidence to support causal association between HDPs and the risks of ovarian, cervical, endometrial, breast cancer, and uterine fibroids.
36,37 A longitudinal study, featuring an average follow-up duration of 19 years, conclude that HDPs did not constitute an independent risk for prevalent female cancers (ovarian, endometrial, and breast). 11However, a nationwide cohort investigation with an average follow-up period of 17.7 years discovered that a history of HDPs correlated with a diminished occurrence of breast cancer and an augmented incidence of endometrial cancer, but had no association with ovarian or cervical cancer. 12This MR analysis, using European ancestry data, determined that genetically predicted HDPs were not associated with gynecologic tumors.Nevertheless, further stratified analysis by race or country is challenging to achieve, which may contribute to the discrepancies between the MR analysis results and those of earlier investigations.A contemporary Nordic research, encompassing 116, 196 breast cancer cases, demonstrated a reduced risk of breast cancer in women with a history of gestational hypertension or preeclampsia, following adjustments for country, maternal birth year, and parity. 38Subsequently, an elevated endometrial cancer risk in Nordic population was observed to be associated with HDPs, including gestational hypertension and preeclampsia. 39Opposite findings regarding the reduction in cancer risk have been documented in studies conducted on populations from Sweden, Norway, Scotland, and the United States. 6,7,10,40,41oreover, an Italian case-control research has identified a significant elevation in the risk of breast cancer, which was associated with a history of gestational hypertension. 42A recent nationwide prospective cohort study involving 40, 720 parous women found no associations between a history of HDPs and risk of breast cancer.However, when the analysis was stratified according to race and ethnicity, a positive association was suggested between HDPs and the risk of breast cancer among Hispanic/Latina women. 13hese varying findings might be attributed to underlying differences in various populations.Consequently, it is plausible that intrinsic genetic diversity among different study populations could lead to disparate outcomes.Further MR analyses are warranted across diverse populations to better understand these relationships.
Most current studies tend to focus on HDPs, especially preeclampsia, and future risks of breast cancer and endometrial cancer.A 2021 meta-analysis deduced that women who have experienced preeclampsia have a 19% reduced risk of developing premenopausal breast cancer. 43This inverse relationship was further substantiated by a combined analysis of six separate cohorts. 36Several theories have been put forth to clarify the observed decrease in breast cancer risk among individuals with a history of HDPs, specifically preeclampsia.The prevailing hypothesis suggests that preeclampsia/eclampsia incites hormonal upheavals and imbalances during pregnancy, which could potentially correlate with the ensuing risk of hormone-dependent cancers. 44The anti-angiogenic profile associated with preeclampsia/eclampsia might confer a protective effect against cancer, considering the necessity of angiogenesis for the growth and metastasis of tumors. 38,45This suggests a potential correlation between preeclampsia/eclampsia and a diminished likelihood of gynecologic tumors later in life.Supporting this hypothesis, a decrease in breast cancer risk has been recorded in women who have a history of preeclampsia/ eclampsia, 44 particularly among premenopausal women and those with breast cancer associated with ERB-B2 receptor tyrosine kinase 2. 6,7,46 However, this association is not universally recognized.Research based on the Norfolk prospective population-based study discovered no association between HDPs and breast cancer. 11Previous Finnish cohort research suggested that the incidence of breast cancer in postmenopausal hypertension patients does not seem to be higher than that in the general population. 47In addition, a recent Taiwanese study also failed to identify an association between HDPs and breast cancer risk, despite a point estimate leaning towards an increased risk. 48y using MR analysis, we can provide a genetic evidence that HDPs does not amplify the risk of subsequent breast cancer.Although our MR analysis was unaffected by confounders, GWAS summary data on breast cancer lacked information on cancer type and stage, which limited our further stratified exploration.Therefore, the present MR analysis cannot provide a comprehensive explanation for the relationship between HDPs and different subtypes and premenopausal or postmenopausal gynecologic tumors.Future research is warranted to further explore the relationships between HDPs and different types and stages of gynecologic tumors.Analogous to breast cancer, the majority of endometrial cancers exhibit hormone-dependency, with key risk factors linked to exposure to endogenous and/or exogenous estrogens. 49Women with preeclampsia tend to exhibit lower estrogen levels and heightened progesterone levels relative to those with normotensive pregnancies. 50dditionally, preeclamptic women may exhibit increased androgen levels, potentially attributable to inadequate placental enzyme production for the aromatization of testosterone to estrogen and elevated inhibin A levels, leading to augmented androgen production. 51Several large prospective studies have suggested a correlation between augmented circulating testosterone concentrations or genetic indicators suggestive of elevated testosterone levels, and a heightened risk of endometrial cancer. 52,53urthermore, the elevated risks related to hypertension and preeclampsia indicated that immunological and inflammatory causes during and prior to pregnancy may also be significant contributors to developing endometrial cancer.Specifically, a connection has been established between preeclampsia and the augmentation of inflammation and immune activation in maternal circulation and the uteroplacental unit, 54 factors that could be relevant to the escalated risk of endometrial cancer.However, epidemiological studies examining the association between preeclampsia and endometrial cancer risk have yielded inconsistent results.For instance, a large Danish casecontrol research discovered no correlation between preeclampsia and the risk of endometrial cancer.However, a stratified analysis revealed an elevated risk of endometrial cancer in cases of early onset preeclampsia, while no correlation was observed in instances of late onset disease. 55he Jerusalem Perinatal Study found no correlation between preeclampsia and uterine cancer. 56While the Jerusalem Perinatal Study did not acquire data pertaining to gestational age, it remains conceivable that a significant number of preeclampsia diagnoses were of late onset.Conversely, a nested case-control study encompassing 10, 924 endometrial cancer instances from four Nordic countries concluded that preeclampsia during pregnancy was markedly linked to an increased risk of endometrial cancer.This correlation was observed similarly for both Type I and Type II endometrial cancer. 39Unlike previous observational studies with inconsistent results, this MR analysis provides reliable evidence to support no association between HDPs and future endometrial cancer risk.Earlyand late-onset preeclampsia may have different effects on endometrial cancer.Further exploring the association between the subtypes of preeclampsia/eclampsia or gestational hypertension and gynecologic tumors is needed.
Few studies reported the association between HDPs and the risks of ovarian and cervical cancer, as well as uterine fibroids.A cohort study using Swedish cancer registry data found no correlation between preeclampsia and cervical cancer. 57Similarly, a prospective populationbased investigation with an average follow-up of 19 years determined no connection between HDPs and ovarian cancer. 11A comprehensive meta-analysis, aggregating 13 studies and incorporating 5, 254, 150 participants, suggested no substantial disparity in the susceptibility to uterus-associated malignancies between women experiencing normal pregnancies and those afflicted with preeclampsia.However, the peril of ovarian cancer was amplified in women experiencing preeclampsia relative to those undergoing normal pregnancies. 43Prior researches have merely suggested a correlation between hypertension and uterine fibroids. 58,59At the commencement of hypertension, angiotensin undergoes hydrolysis to form angiotensin I, which is subsequently transformed into angiotensin II via the action of the angiotensin converting enzyme. 60Research has indicated that angiotensin II notably escalates the population of uterine leiomyoma cells, with this effect being proportionate to the dosage. 61 study conducted by Hsieh et al. discovered a significant correlation between mutations in genes responsible for activating angiotensin-converting enzyme and susceptibility to leiomyoma. 62These findings suggested that the production of angiotensin II, as a result of hypertension, could potentially trigger uterine leiomyoma.Further, hypertension may stimulate the proliferation of fibroids and fibrogenesis by causing injury to smooth muscle cells via mechanical shear stress, which could also contribute to the development of uterine fibroids. 63Nevertheless, there are currently no reports on the connection between HDPs and the subsequent risk of developing uterine fibroids.The recent MR study have shown that higher genetically predicted SBP and diastolic blood pressure (DBP) were related to an increased risk of uterine fibroids. 64To our knowledge, this is the first MR study to report the association between HDPs and ovarian cancer, cervical cancer, and uterine fibroids.The genetic prediction that HDPs are not associated with ovarian cancer, cervical cancer, and uterine fibroids may provide valuable insights for future research directions: (1) population-or MR-based studies are needed to further explore the association of HDPs with ovarian cancer, cervical cancer, and uterine fibroids in non-European populations, such as East Asian and African populations; (2) the association between HDPs and subtypes and stages of various gynecologic tumors needs further research and validation.
However, several limitations within this study are worth considering.First, our findings are derived from data of European ancestry in an effort to control for racial effects, which consequently limits their applicability to non-European populations.Second, potential sample overlap may exist given that both exposure and outcome datasets originate from European populations.Unfortunately, the evaluation of overlapping sample sizes continues to present a challenge.The presence of overlapping samples may bias the estimations derived from a two-sample MR study.Recent research posited that two-sample MR approach can be reliably employed when dealing with large data from a single source like UK Biobank. 65Although our findings indicated no association between HDPs and common gynecologic tumors, it needs to be verified in further MR studies.Third, the restriction of GWAS summary data on HDPs and gynecologic tumors makes it challenging to stratify research based on the current results, an issue that warrants attention upon future dataset updates.

F I G U R E 2
Causal association between any hypertensive disorders of pregnancy (HDPs) and common gynecologic tumors.| 7of 16ZHOU et al.

F I G U R E 3
Causal association between gestational hypertension and common gynecologic tumors.T A B L E 4 Univariate Mendelian randomization (UVMR) analysis of gestational hypertension with common gynecologic tumors in European population.

1
Study design diagram and three assumptions of Mendelian randomization (MR).Summary of the genome-wide association studies (GWAS) included in this MR study.

Trait Dataset Sample size Number of SNPs Population Consortium Sex Year
Abbreviation: HDPs, hypertensive disorders of pregnancy.
Univariate Mendelian randomization (UVMR) analysis of hypertensive disorders of pregnancy (HDPs) with common gynecologic tumors in European population.Multivariable Mendelian randomization (MVMR) analysis of hypertensive disorders of pregnancy with common gynecologic tumors in European population.
Causal association between preeclampsia/eclampsia and common gynecologic tumors.Univariate Mendelian randomization (UVMR) analysis of preeclampsia/eclampsia with common gynecologic tumors in European population.
F I G U R E 4